Investigations in the past decades have significantly advanced our understanding of signaling mechanisms underlying the protection and pathogenesis of myocardial ischemic injury. It is increasingly recognized that preservation of mitochondrial function plays a pivotal role in cardioprotection against ischemia reperfusion injury (I/R). However, it remains virtually unknown as to who the molecular targets of cardioprotection are in the mitochondria;what specific molecular events led to the protection of mitochondria;and whether there is a systems integration of cardioprotective signaling at the mitochondria to support the manifestation of a protected phenotype. Using a murine model of nitric oxide (NO) induced late phase of cardioprotection, we elect to examine the plausible intrinsic signaling properties of mitochondria using a novel experimental strategy enabling a parallel examination of mitochondrial signaling, mitochondrial proteomes, and mitochondrial behavior by computational modeling. The proposed studies are based upon preliminary evidence by others and our own demonstrating that activation of PKC?-Src module occurs in the NO donor treated mice and that both are localized to mitochondrial membranes. In this proposal we will test the innovative hypothesis that the PKC?-Src module interacts with the brief mPTP openings to protect cardiomyocytes from Ca++ overload induced jury. The working hypothesis is that NO activates PKC?-Src module, leading to brief mPTP openings which results in transients Ca++ releases and reactive oxygen species (ROS) bursts, and consequently inactivates Ca++ reuptake and further activates PKC?-Src to form a feed- forward loop. When the homeostasis is interrupted (e.g., calcium overload or elevated ROS), brief mPTP openings transits into irreversible, long-lasting mPTP openings, which instead induce cardiac injury. In this application we propose to delineate the functional effects of brief openings of mPTP on Ca++ handling and ROS production;and to elucidate mPTP regulation by the mitochondrial PKC?-Src module in the setting of NO-induced late phase of cardioprotection (Aim 1). In the specific Aim 2 we will conclusively establish the activation of a PKC?-Src signaling module in the mitochondria as a mandatory signaling element of NO-induced cardioprotection against myocardial ischemic injury. At last we will systematically define the molecular targets of mitochondrial Src-kinase in NO- induced late phase of cardioprotection (Aim 3). The proposed studies will advance our understanding of cardiac biology by providing novel mechanistic insights into how interactions of brief mPTP openings with mitochondrial PKC?-Src module can be beneficial in mediating NO-induced late phase of cardioprotection. (End of Abstract)